Fluorescent probe and Raman spectroscopic investigation of the effects of pH, heating, and kappa-carrageenan on whey protein structure

Surface hydrophobicity (S_o) is an important structural parameter describing food proteins due to its correlation with functionality. A new method to measure S_o was established based on the neutral fluorescent probe, 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN).; S_o was determined for whey protein isolate (WPI), bovine serum albumin (BSA) and β-lactoglobulin (BLG) at various pH (3.0, 5.0, 7.0 and 9.0) under heated (80°C for 30 min) or unheated conditions. S _o values measured by PRODAN and cis-parinaric acid (CPA) were lower at pH 3.0 than at other pH, while values measured by 1-anilinonaphthalene-8-sulfonic acid (ANS) were highest at pH 3.0. The anionic probes ANS and CPA yielded opposing results on the effects of pH and heating on protein hydrophobicity.; Heating, pH and addition of κ-carrageenan (KCG) at low (1.0:62.5), medium (1.0:6.0) or high (1.0:1.2) ratios of KCG:protein, as well as the interactions between these factors, significantly (P < 0.05) affected S_o measured by PRODAN. Generally, proteins had higher S_o and were more sensitive to heating and KCG addition at pH 9.0, than at other pH. Heating increased S_o of high ratio KCG:protein mixtures at pH 3.0, but generally decreased S_o of mixtures at higher pH, especially pH 9.0.; Raman spectroscopy was used to analyze structural changes at higher protein concentration (15% w/v) than those (0.002–0.01%) used in spectrofluorometry. Decreased SS and Trp band intensities, and lower helical and higher β-sheet content, were generally observed after heating at pH 9.0 or KCG addition at pH 5.0. Decreased helical and increased β-sheet contents obtained by heating BLG at pH 7.0 or 9.0 were not observed after heating KCG:BLG mixtures at these pH. Addition of KCG to BSA at pH 7.0 or 9.0 resulted in increased helical content. Heating KCG:WPI mixtures at pH 5.0 and 9.0 resulted in large increases in SS and Trp band intensities and helical content, and decreased ratio of the tyrosine doublet, indicating a more buried or hydrophobic environment around aromatic residues.; Raman and fluorescent probe spectroscopy provided information on protein structural properties as a function of pH, heating and interactions with other macromolecules, which may be important in applications of these proteins in food systems.